HIV-1 structural protein Gag drives virus particle formation primarily at the plasma membrane. The plasma membrane consists of microdomains containing different sets of lipids and proteins. Microdomains such as lipid rafts and tetraspanin-enriched microdomains (TEMs) are highly dynamic and can be stabilized by oligomerization of associated proteins. Lipid rafts and TEMs are implicated in multiple cellular functions including signaling and trafficking, often as delivery platforms. Notably, HIV-1 assembly occurs at the plasma membrane region enriched with these microdomains. As Gag binds to and multimerizes beneath the plasma membrane cytoplasmic leaflet, Gag may stabilize lipid rafts or TEMs at virus assembly sites. Although lipid rafts are shown to facilitate HIV-1 particle assembly, it is unknown whether HIV-1 assembly alters the plasma membrane organization and whether such alteration affects steps following virus assembly. The goal of this proposal is to test our central hypothesis that Gag multimerization at the plasma membrane reorganizes microdomains, which in turn affects post-assembly steps such as cell-to-cell transfer of virions. Our specific aims are: [Aim 1] To examine whether Gag membrane binding and multimerization alter microdomain organization of the plasma membrane. We will elucidate requirements for recruitment of two distinct groups of microdomains, lipid rafts and TEMs, to assembly sites using quantitative microscopy techniques. We will analyze association of microdomains with different stages of Gag multimers using correlative FRET/scanning electron microscopy. Temporal aspects of microdomain organization will be examined using total internal reflection fluorescence microscopy. Analyses of Gag derivatives using these techniques will help us understand microdomain dynamics at virus assembly sites. [Aim 2] To determine the role of Gag multimerization in virological synapse (VS) formation. VS is a raft- and TEM-enriched contact structure formed between a virus-producing T cell and a non-infected T cell, which mediates efficient transfer of virus particles. Preliminary data suggest that Gag accumulates to a rear-end protrusion of T cells known as uropod in a multimerization-dependent manner and that the Gag-laden uropod serves as a precursor of VS. Roles for Gag multimerization and microdomain association in uropod localization will be elucidated using fixed and live cell microscopy. Altogether, experiments in this proposal will allow us to understand relationships between Gag multimerization and microdomain organization and help us identify a new strategy to suppress spread of HIV-1, the causative agent of AIDS. PUBLIC HEALTH RELEVANCE: The goals of the proposed research are to determine the effects of HIV-1 particle assembly on the plasma membrane microdomains and to elucidate the consequence of such effects in virus spread. Knowledge obtained from proposed studies will help us identify novel targets for antiretroviral strategies that suppress virus spread, a key aspect of HIV-1 pathogenesis